Abstract:

Mechanisms nourish stem cells for organ regeneration and prevent alcohol
related diseases such as Fetal Alcohol Syndrome (FAS) and Liver
Sclerosis. These stem cell nutrients have been found to positively affect
the skin, liver, brain neurons, pancreas, and the GI tract. Cholesterol
supplementation prevents fetal alcohol spectrum defects (FASD) in
alcohol-exposed zebra fish embryos. Using the zebra fish model, alcohol
was found to interfere with embryonic development by disrupting
cholesterol-dependent activation of a critical signaling molecule, sonic
hedgehog (Shh). Cholesterol supplementation of the alcohol-exposed
embryos restored the functionality of the molecular pathway and prevented
development of FASD-like defects. Novel biomarkers were identified for
diagnosing alcohol related diseases by lipid chemical analysis and Raman
Spectroscope.

Claims:

1. A method of detecting the presence of alcohol or cholesterol lowering
components and damage associated therewith in embryonic and adult tissue
and organs comprising determining the level of defectiveness which has
occurred to Shh protein in the tissue and organs due to the alcoholic or
cholesterol lowering components.

3. A method for reducing a condition associated with fetal alcohol
syndrome in a subject exposed to alcohol in utero, the method
comprising:administering the subject a cholesterol or a cholesterol
derivative in an amount sufficient to reduce the condition associated
with fetal alcohol syndrome.

4. A method for screening and identifying one or more agents which are
protective or therapeutic for fetal alcohol syndrome and adult stem cell
aging related defects, comprising:administering the agent to a zebra fish
embryo model or rat hepatic stellate cell lines before, after or
concurrently with the transient alcohol exposure of the embryo;
anddetecting a serial of molecular and cellular defects in the alcohol
treated embryo compared to a control.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]Not applicable

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002]Not applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

[0003]Not applicable

BACKGROUND OF THE INVENTION

[0004]1. Field of the Invention

[0005]This invention relates generally to a breakthrough in formation and
rejuvenation of organs through stem cell nutrients and alcohol damaged
organ regeneration. New mechanisms have been discovered which nourish
stem cells for organ regeneration and prevent alcohol related diseases
such as Fetal Alcohol Syndrome (FAS) and Liver Sclerosis. These stem cell
nutrients have been found to positively affect the skin, liver, brain
neurons, pancreas, and the GI tract.

[0006]2. Description of Related Art

[0007]Consumption of alcohol by pregnant women can cause fetal alcohol
spectrum defects (FASD), a congenital disease, which is characterized by
an array of developmental defects that include neurological,
craniofacial, cardiac, and limb malformations, as well as generalized
growth retardation. FASD remains a significant clinical challenge and an
important social problem. Although there has been great progress in
delineating the mechanisms contributing to alcohol-induced birth defects,
gaps in our knowledge still remain; for instance, why does alcohol
preferentially induce a spectrum of defects in specific organs and why is
the spectrum of defects reproducible and predictable.

[0008]Alcohol related birth defects leave around 100 babies every day in
United States alone with little chance of having more than average IQ,
and many with some malformed organs. The cost to the US for the care of
these children is staggering at an estimated annual cost of $10 billion
to the health care system. Fetal alcohol syndrome is a term used to
describe an array of developmental defects affecting the nervous and
cardiovascular systems. The syndrome also can lead to growth retardation,
facial abnormalities and lowered mental functioning.

[0009]The keys to fetal alcohol syndrome's severity are the amount of
alcohol consumed, the duration of the consumption and the timing of the
pregnancy. For example, alcohol consumed by a mother with a one-month-old
fetus could alter the development of the brain; at four to eight weeks,
facial structures, heart and eyesight could be affected. Two to three
months into fetal development, alcohol consumption could lead to the
growth of extra digits. The amount of alcohol consumed is important as
well. Even the equivalent of one 12-ounce beer, consumed at the wrong
time, could disrupt the signaling pathway and lead to a defect. Increased
amounts of alcohol exposure by the fetus lead to increased severity of
the syndrome.

BRIEF SUMMARY OF THE INVENTION

Fetal Alcohol Syndrome

[0010]Applicant has found that cholesterol supplementation prevents fetal
alcohol spectrum defects in alcohol-exposed zebra fish embryos. Using the
zebra fish model, applicant has found alcohol interferes with embryonic
development by disrupting cholesterol-dependent activation of a critical
signaling molecule, called the sonic hedgehog (Shh). Cholesterol
supplementation of the alcohol-exposed embryos restored the functionality
of the molecular pathway and prevented development of such defects.
Alcohol related-like defects in zebra fish resulted from minimal fetal
alcohol exposure, equivalent to a 120-pound woman drinking one 12-ounce
bottle of beer. The findings suggest even small amounts of alcohol might
be unsafe for a pregnant woman and also indicate cholesterol
supplementation may be a potential means of preventing fetal alcohol
defects.

[0011]Small amounts of alcohol can interfere with the growth of a fetus,
but added cholesterol may help prevent a wide array of neurological and
physical defects from alcohol exposure. Cholesterol is so important to
fetal development that pregnant women who do not have physiological high
enough cholesterol levels are at increased risk of having babies with
developmental problems, even without consuming alcohol. Alcohol, even in
small amounts, blocks the ability of cholesterol to orchestrate the
complex series of events involved in regulating cell fates and organ
development in the embryo. Encouragingly, giving supplemental cholesterol
to zebra fish embryos exposed to alcohol restored normal development.

[0012]Alcohol interferes with a precisely orchestrated biochemical
signaling pathway that guides fetal development. Cholesterol is essential
for a single pathway that governs the pattern of tissue development and
it is vulnerable to the effects of alcohol. This new insight into the
molecular basis of fetal alcohol syndrome could have far-reaching
implications and suggests new prenatal care that might prevent the
developmental defects caused by alcohol consumed during pregnancy.

Adult Organ Rejuvenation

[0013]Giving alcoholics supplemental cholesterol may help slow down or
prevent the occurrence of alcoholic liver disease, even chronic alcoholic
induced cirrhosis, characterized by replacement of liver tissue by scar
tissue, leading to progressive loss of liver function. The findings
provide further credence to current practice of ensuring that pregnant
women should not lower their cholesterol too low. A recent study found
that women who took cholesterol-lowering drugs known as statins were at
greater risk of giving birth to babies with developmental problems.

[0014]This new concept, stem cell nutrient and related technology, is also
monumental for leading adult stem cell based healthcare and clinical
practice in the coming years. This disclosed technology applied to adult
health will have a major impact in anti-aging, organ and tissue
regeneration, and prevention of alcohol related diseases.

[0015]Stem cell nutrients are foods that have both prescription and over
the counter applicability. Thus the markets generally are:
[0016]Vitamin Supplements [0017]Supplements aimed as Specific Organs
[0018]Nutritional Food Additive [0019]Prescription Drug with a Variety of
Delivery Methods [0020]Supplement for Women subject to Pregnancy

[0021]The following embodiments and aspects thereof are described and
illustrated in conjunction with systems, tools and methods which are
meant to be exemplary and illustrative and not limiting in scope. In
various embodiments one or more of the above-described problems have been
reduced or eliminated while other embodiments are directed to other
improvements. In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become apparent by
reference of the drawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0022]The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings(s) will be provided by the Office upon
request and payment of the necessary fee.

[0023]FIG. 1 graphically demonstrates dose-dependent effects of alcohol on
the survival and phenotype of embryos.

[0024]FIG. 2 shows photocopies of the effects of fetal alcohol exposure
which induces a phenotype spectrum similar to that of Hh-inhibited and
cholesterol deficient embryos.

[0044]Exemplary embodiments are illustrated in reference figures of the
drawings. It is intended that the embodiments and figures disclosed
herein are to be considered to be illustrative rather than limiting.

DETAILED DESCRIPTION OF THE INVENTION

Cholesterol Treatment for Fetal Alcohol Syndrome

[0045]In this application, exposure of zebra fish embryos to low levels of
alcohol during gastrulation blocks covalent modification of Sonic
hedgehog by cholesterol. This leads to impaired Hh signal transduction
and results in a dose-dependent spectrum of permanent developmental
defects that closely resemble FASD. Furthermore, supplementing
alcohol-exposed embryos with cholesterol rescues the loss of Shh signal
transduction, and prevents embryos from developing FASD-like morphologic
defects. Overall, a simple post-translational modification defect in a
key morphogen may contribute to an environmentally induced complex
congenital syndrome. This insight into FASD pathogenesis may suggest
novel strategies for preventing these common congenital defects.

[0046]Post-translational protein modification plays an essential role in
facilitating signal transduction regulation of gene expression. Protein
modification by phosphorylation, acetylation, or methylation helps
control the proper timing and sequence of events during embryogenesis;
therefore, it is not surprising that defective modifications of these
proteins can be important causes in the development of many types of
congenital diseases. Accumulating evidence illustrates the importance of
post-translational lipid modifications for regulating protein function.
One example is the cholesterol and palinitoyl modification of Sonic
hedgehog (Shh), which guides this protein's biogenesis, cellular
trafficking, and functionality.1

[0047]Shh is a highly conserved fetal morphogen that plays a central role
in cell proliferation, differentiation, and embryonic patterning by
activating the Hedgehog (Hh) signal pathway.2,3 The 45 kDa Shh
precursor protein undergoes modification by auto-processing, followed by
covalent linkage of cholesterol to the N-terminal proteolytic
product.4 This mature, cholesterol-modified protein (19 kDa) can be
transported to the cell membrane for secretion.5 Once secreted, the
cholesterol-modified Shh ligand can initiate signal transduction by
binding to its receptor, Patched (Ptc). Upon binding, Ptc relieves the
inhibition of the signal transducer, Smoothened (Smo)6, which then
activates Gli transcription factors by uncoupling them from the negative
regulator, Suppressor of Fused.7 Gli is subsequently translocated to
the nucleus and regulate expression of target genes including Ptc8,
Gli19 itself and NkX2.2.10

[0048]During embryogenesis, Shh is expressed specifically in Hensen's
node, the floor plate of the neural tube, the cardiac mesenchyme, the
early gut endoderm, the posterior portion of the limb buds, and
throughout the notochord. As it is a morphogen, Shh also affects the
development of tissues that are distal to where it is produced. Shh is
apparently a key inductive signal for patterning of the ventral neural
tube11,12, the anterior-posterior limb axis13, and the ventral
somites.14 In humans, one severe phenotype caused by mutations in
Shh, or other components of Hh signaling, is holoprosencephaly
(HPE)15, a disorder in which the fetal prosencephalon (forebrain)
fails to divide to allow formation of bilateral cerebral hemispheres. HPE
is also one of the extreme manifestations of severe fetal alcohol
spectrum defects (FASD) in human embryos and in animal models of
FASD15-20. Similarly, production of Shh in the floor plate of the
neural tube regulates the development of neural components in the
overlying basal plate, including progenitors of motor neurons12,21.
FASD patients display delayed motor development and impaired fine- and
gross-motor skills.22 Varying degrees of motor retardation have been
observed in up to 89% of humans having FASD.23-25 Indeed, the
diagnostic criteria for FASD include impaired fine motor skills.26
Shh also has a proven role in neural crest morphogenesis27, and FASD
frequently includes defects in neural crest-derived structures. Clearly,
there is significant overlap between the tissues affected by alcohol
exposure, and those tissues that depend on Shh signaling for proper
development.

[0049]Post-translational modification of Shh by cholesterol4 is a
tightly regulated process that is necessary for the transportation and
establishment of concentration gradients of the mature Shh ligand in
developing embryos28. Sterol- and fatty acid-modified Shh proteins
form soluble multimers that are packaged in micelles for long-range
transport.29 Recent work has demonstrated that the activity and
function of Shh protein varies significantly, depending upon the presence
or absence of these modifications.5 The roles played by
cholesterol-modified Still ligand in many facets of embryogenesis may
account for some of the teratogenic effects of perturbed cholesterol
biosynthesis in animal development.30 Similar congenital defects
occur in offspring of women who drink alcohol during pregnancy.

[0050]The teratogenic consequences of fetal exposure to alcohol are highly
variable and include a spectrum of morphologic defects known as
FASD.31 Phenotypic abnormalities of FASD include neurological,
craniofacial, cardiac, and limb malformations, as well as generalized
growth deficits and mental retardation.32 The mechanisms proposed to
underlie the spectrum of birth defects caused by fetal alcohol exposure
include: apoptosis33, cell adhesion defects34, accumulation of
free radicals35, dysregulation of growth factors38, and altered
retinoic acid biosynthesis37. Some simple and essential questions
have not been well explained by these hypotheses, for instance, how one
or, at most, a few social drinks, cause fetal defects, why alcohol
preferentially induces defects targeting some organs and tissues and not
others, or why the pattern of defects seen in FASD is predictable and
reproducible.

[0051]Alcohol can also impair prechordal plate migration38 and
disrupt the formation and function of Spernann's Organizer39, a
signaling center in gastrulating embryos that controls the patterning of
the germ layers; the specific mechanisms that regulate axis pattern
formation require highly evolutionarily conserved genetic pathways
involving transcription regulatory circuitry and signal transduction
pathways40 Shh-containing vesicles contained within the organizer
initiate a signal transduction pathway that plays a key role in embryonic
patterning during development.41 Therefore, genetic or environmental
factors that inhibit Shh signaling during gastrulation can disrupt proper
patterning of the embryo. Interestingly, embryos that are exposed to
alcohol during gastrulation38 have defects that are similar to those
found in embryos that have defects in Hh signal transduction42. A
similar phenotype develops in embryos with a genetic defect in sterol
homeostasis, for example, Smith-Lemli-Opitz Syndrome43 (SLOS) or
that are exposed to cholesterol lowering agents.44.45 These
observations suggest that FASD may result from alcohol-dependent
inhibition of cholesterol modification of Shh.

[0052]Many genetic disorders can result in abnormal regulation of
cholesterol biosynthesis, storage, and trafficking. However, ethanol
ingestion may be far more common mechanism for disrupting cholesterol
homeostasis. Ethanol causes an inhibition of HMG-CoA reductase activity,
which results in decreased free cholesterol in the cells, and reduction
in circulating cholesterol levels.46-48 Acute ethanol exposure in
perfused rat liver results in depletion of cholesterol in both liver
homogenate and microsomes.49 Ethanol specifically inhibits hepatic
ACAT activity, which can lead to decreased cholesterol esters for
transport in LDLS.50 Thus, evidence from embryology, toxicology, and
molecular biology indicates that a teratogenic mechanism underlying FASD
links alcohol, cholesterol homeostasis, Shh signaling and cholesterol
modification of functional Shh.

[0053]Several animal models have been to study FASD. The zebra fish model
offers many advantages compared to insect and rodent models for alcohol
and development studies: zebra fish are small in size, they have a large
number of progeny, and they have rapid embryogenesis. This model has
already been widely used in studies of developmental biology, genetics,
gene function, signal transduction and high throughput drug screening.
All of these characteristics make it an ideal model to delineate the
molecular basis of the alcohol-induced birth defects.

[0054]It is shown that transient alcohol exposure during early development
of zebra fish embryos causes dose-related inhibition of Hh signal
transduction and produces a spectrum of permanent FASD-like defects.
Alcohol-induced inhibition of Hh pathway activity parallels alcohol
disruption of cholesterol homeostasis and decreased
cholesterol-modification of the Shh ligand. Supplementing the
alcohol-exposed embryos with cholesterol rescues the loss of Shll signal
transduction, and prevents embryos from developing FASD-like morphologic
defects.

Materials and Methods

Alcohol, Cyclopamine, and AY-9944 Treatment

[0055]Embryonic alcohol exposures were adapted from a previous
report.38 Embryos with chorions were exposed to six different
concentrations of alcohol (eg, 0, 0.25, 0.5, 1.0, 1.5, and 2.0% (v
v-1)) in embryo medium. Embryos in sealed Petri dishes were exposed
to alcohol for 6 h beginning at the dome stage (ie, 4.25 hours
post-fertilization (hpf) or 30% epiboly stage) and were incubated at
28.5° C. Immediately following alcohol exposure, embryos were
harvested for analysis of Hh pathway activity, cholesterol content, or
tissue alcohol concentration. The remaining embryos were washed and
incubated in alcohol-free medium. Embryos were then harvested at 1, 2, or
4 days post-fertilization for survival and phenotypic analyses.
Cyclopamine (11-deoxojervine) is a naturally occurring chemical that
inhibits the Hh signaling pathway by functioning as an antagonist of
smoothened protein. AY9944, trans-1,4 bis-(2-dichlorobenzylaminomethyl)
cyclohexane dihydrochloride blocks cholesterol synthesis through
inhibition of 7-dehydrocholesterol reductase. AY-9944 (7.5 μM,
Sigma-Aldrich) and cycloparnine (10.0 μM, Calbiochenn) were
administered in the same manner as alcohol. Following treatment, embryos
were washed and incubated in normal medium for up to 4 days
post-fertilization.

Alcian Blue Staining and Immunohistochemistry

[0056]Staining for skeletal structures was performed as previously
described.51 Immunohistochemistry is carried out with following
primary antibodies (Hybridoma Bank, 1:10): MF20 to stain myocardium and
facial muscles, S46 to identify ventricular myocardium, and F59 to
identify slow muscle progenitors in the somites. The secondary antibodies
were Alexa 568-conjugated goat anti-mouse IgG2g and Alexa
488-conjugated goat anti-mouse IgG2g (1:400, Molecular Probes). The
embryos were mounted and imaged.

[0058]Following alcohol exposure, embryos (n=38) were pooled from each
treatment group in triplicate. An Alcohol Test Kit (Diagnostic Chemicals
Limited) was used to determine the tissue alcohol concentrations in
treated embryos.

RT-PCR and Real-Time Quantitative Analysis

[0059]Total RNA was extracted from embryos (n=10) with RNeasy kits
(Qiagen). RT-PCR were performed using primers (information listed in
following table) as previously described.52

[0061]The Gli-luciferase reporter assay was performed in replicate
experiments of pooled embryos (n=15). Briefly, zebra fish embryos at the
1-2 cell stage were microinjected with 0.5 nl of Gli-BS-Firefly
luciferase plasmid (60 ng nl-1) and Renilla luciferase plasmid (60
ng nl-1, pRL-TK, Promega). Reporter activity was determined by using
the Dual-Luciferase Reporter Assay System (Promega). Activity of the
Firefly luciferase reporter was normalized to the activity of a Renilla
luciferase internal control for transfection efficiency.

Cholesterol Microinjection

[0062]Embryos were microinjected at 1-2 cell stage with 0.2 nl of 5 μg
μl-1 (10 pg) cholesterol (BioVision Inc.) with or without the two
plasmids for the Gli-luciferase reporter assay. Embryos were allowed to
develop and were then treated with alcohol as previously described. At 48
hpf embryos were analyzed.

Results

Alcohol Exerts Teratogenic Effects in a Dose and Stage-Specific Manner

[0063]The zebra fish model was chosen to evaluate the hypothesis because
it permits exposure to precise concentrations of alcohol during
well-defined developmental time frames. Zebra fish embryos were exposed
to a range of alcohol concentrations (0, 0.25, 0.5, 1.0, 1.5, and 2.0%
v/v in embryo medium at two different time windows during development.
The first exposure window occurs from 1 to 2 cell stages to 3 hours post
fertilization (hpf), and the second exposure window occurs between 4.25
and 10.25 hpf during the late blastula stage and the gastrula stage.
Exposure to alcohol during the first exposure window is almost uniformly
fatal. Fewer than 40% of the 897 embryos from this time frame that were
treated with the lowest alcohol concentration (0.25%) survived to 48 hpf.

[0064]Embryos exposed to alcohol during the second exposure window had
much better survival rates than those exposed during the zygote stage to
the same levels of alcohol. During the late blastula-gastrula stage,
survival of the exposed embryos was also dose dependent. For example, 10%
of 202 embryos exposed to 3% alcohol during this time frame were alive at
48 hpf, compared to a survival rate of over 90% for the 897 embryos
exposed to 0.25% alcohol for 6 h during the same developmental time
frame. A more detailed analysis was performed at 48 hpf by scoring
alcohol effects in three categories: (a) dead, (b) alive with abnormal
phenotype, or (c) alive without abnormal phenotype.

[0065]As shown in FIG. 1A, during the late blastula-gastrula stage, the
phenotype at 48 hpf depended upon the dose of alcohol that embryos were
exposed to. For example, 84% of the embryos exposed to 2% alcohol during
the second exposure window survived through 48 hpf and exhibited abnormal
morphology, while only 2.6% of the living embryos were phenotypically
normal. (See FIG. 2 for illustrations of representative defects). This
level of exposure was lethal for the remaining 13% of embryos. In
contrast, 18% of the embryos exposed to 0.25% alcohol during the second
exposure window were alive and had minimal abnormalities at 48 hpf; the
majority (72.3%) were alive and had normal phenotypes and <10% failed
to survive to the 48 hpf time point. The frequency of these
alcohol-induced phenotypes has been characterized in Table 1.

[0067]To assure that the alcohol-induced morphologic defects seen in our
model do not merely reflect exposure to supraphysiologic concentrations
of alcohol, applicant measured the alcohol concentrations in fetal tissue
following alcohol exposures. Tissue alcohol concentrations in zebra fish
embryos correspond to the exposed alcohol concentration in embryo medium
0.25-2.0% range from 0.71-7.4 mM or 0.003-0.034 g dl-1. (FIG. 1B)
These alcohol concentrations can be achieved in the blood of a human
being by consumption of one or, at most, a few social drinks. As shown in
FIG. 1, embryos (n>64) were exposed to increasing concentrations of
alcohol and evaluated at 48 hpf. Embryos were scored as alive/normal,
alive/abnormal, or dead. These three categories are expressed as a
percentage of the total number of embryos in each cohort. Tissue alcohol
concentrations in zebra fish are related to the level of alcohol
exposure. In general, the alcohol concentrations were in the range of
0.71-7.4 mM or 0.003-0.034 g dl--1. Error bars indicate 1 s.e.m. of
three experiments (n=38 fish per group).

[0068]Alcohol-induced defects in zebra fish embryos recapitulated those
that occurred in other species following fetal alcohol exposure. In a
dose-dependent fashion, transient alcohol exposure for 6 h during
gastrulation resulted in subsequent permanent phenotypic abnormalities
while only a modest increase in the rate of embryo mortality was observed
(FIG. 1A). By 48 hpf, a cumulative cranial-to-caudal phenotype was
evident in embryos that were exposed to alcohol transiently during
gastrulation (FIG. 2).

[0069]These embryos were growth retarded (FIG. 2A), and exhibited a
dose-dependent spectrum of phenotypes that included neurological,
craniofacial, cardiac, and body axis defects. Embryos exposed to the
highest alcohol concentrations had overt HPE, cyclopia (complete or
partial), pericardial edema, U-shaped somites and severely foreshortened
tails (FIGS. 2A and 2B). Comparisons performed between alcohol-treated
embryos and embryos transiently treated with cyclopamine, a specific
inhibitor of Hh signaling, or AY-9944, an inhibitor of cholesterol
biosynthesis and transportation, during gastrulation revealed that
embryos from all three groups had HPE and partial cyclopia, as well as
underdevelopment of the craniofacial bones and muscles, and failure of
the heart tube to loop (FIG. 2C). These observations support a role for
defective Shh signaling in the pathogenesis of FASD and are consistent
with the possibility that alcohol inhibits Shh activity by interfering
with cholesterol modification of the cleaved 19 kDa protein.

[0070]To investigate this further, Shh signaling activity was directly
measured by examining developing embryos that were microinjected with a
Shh-responsive, Gli-BS luciferase reporter construct at the 1-2 cell
stage53. Following alcohol exposure, both luciferase activity (FIG.
3A) and the expression of Shh-regulated genes, such as Ptc, Gli1, and
Nkx2.2 (FIG. 3B), decreased in a dose-related fashion. Real-time
quantitative PCR analysis confirmed that there is a threshold for
alcohol-induced inhibition of the expression of Shh target genes. While
exposure to a very low alcohol concentration (0.25%) caused no
significant change in the expression of Ptc or Gli1, Nkx2.2 expression
decreased by about 50% under these conditions. In response to a 0.5%
alcohol treatment, expression of these three Shh target genes decreased
by from 1.3 to 1.9 fold, and alcohol concentrations ranging from 1.0 to
2.0% caused expression of those genes to decrease from 5 to 17 fold (FIG.
3C). In contrast to the results obtained by RT-PCR, real-time
quantitative PCR showed that Shh transcription decreased when embryos
were exposed to alcohol concentrations higher than 1.0%. Notably,
inhibition of Shh signaling occurred despite relatively stable levels of
Shh protein from whole cell lysates (cytosolic and membrane). However,
inhibition of Shh signaling was associated with a progressive loss of Shh
from the cellular membrane protein fraction isolated from FASD embryos
(FIG. 3D). Given that Shh must be covalently modified by cholesterol to
anchor in plasma membranes, these results suggest that cholesterol
modification of Shh may be impaired in alcohol-treated embryos.

[0071]Alcohol exposures inhibit Hh signaling by decreasing the
post-translational cholesterol modification of Shh. (a) As seen in FIG.
3, dose-related reduction of a Hh-responsive Gli-luciferase activity
(normalized by Renilla luciferase) in alcohol-exposed embryos. Error bars
indicate 1 s.e.m. of four replicate experiments. (b) RT-PCR analysis of
gene expression levels of Shh, and its target genes, Ptc, Gli1, and
Nkx2.2, as Well as the GAPDH housekeeping gene following alcohol
exposure. (c) Semi-quantitative expression analysis of Shh and its target
genes by real-time RT-PCR, data is normalized to the internal control of
GAPDH. (d) Western blot of the total (cytosolic and membrane) and
membrane fractions of Shh protein from alcohol-exposed embryos.
β-Actin was employed as the loading control.

[0072]As shown in FIG. 3, alcohol exposure during the late
blastula-gastrula stage causes a dose-dependent reduction in membrane
associated Shh. Given that esterification of Shh by cholesterol drives
its membrane localization, these results also suggest that alcohol
exposure reduces cholesterol ester formation. Applicant tested whether
alcohol exposure impairs general sterol homeostasis during gastrulation
by measuring cholesterol levels in whole embryo extracts. In a
dose-related fashion, alcohol exposure resulted in a decrease in the
total cholesterol content of the embryos (FIG. 4). As seen in FIG. 4,
alcohol exposures decrease cholesterol levels in embryos. Dose-related
reduction of total cholesterol and cholesterol ester levels in
alcohol-exposed embryos. Error bars indicate 1 s.d. This was mostly due a
reduction in total cholesterol esters that paralleled the dose-dependent
decreases in cholesterol-modified Shh protein and Shh signaling activity
(FIGS. 2A and 2B), and correlated with a dose-dependent acquisition of
alcohol-induced morphologic defects: (FIG. 2C) the higher the dose the
more severe and extensive the defects. Together with evidence that
AY-9944 lowers cholesterol and produces similar defects, our findings
suggest that alcohol interrupts cholesterol homeostasis and that depleted
stores of cholesterol results in impaired cholesterol modification of
Shh, leading to decreased Shh signaling, which causes a FASD-like
phenotype.

[0074]To confirm the importance of this potential molecular mechanism for
FASD, applicant performed rescue experiments in alcohol-exposed embryos.
Supplemental cholesterol, Gli-BS-Firefly luciferase plasmid, and Renilla
luciferase plasmid were co-microinjected into 1-2 cell stage embryos,
which were then treated with various alcohol concentrations during
gastrulation.

[0075]Subsequent studies showed that Gli-BS reporter activity was
preserved at all doses of alcohol exposure in cholesterol-supplemented
embryos (FIG. 5A). To determine whether recovery of Shh activity leads to
the rescue of Hh-dependent cell differentiation, applicant studied the
slow muscle pioneer (MP) cells in somites42 using the F59 antibody
which specifically identifies these progenitors.54 In untreated
embryos, at 48 hpf, applicant observed an organized, V-shaped pattern of
MP fibers with approximately 20 fibers per somite pair. Microinjection of
5% DMSO (the cholesterol dissolving vehicle) had no adverse effect on
zebra fish development. In contrast, alcohol-exposed embryos had a
disorganized, diffuse pattern of MP fibers at this time. The cholesterol
supplemented, alcohol-exposed embryos had a similar number and pattern of
MP fibers as the untreated, wild-type embryos (FIG. 5B). Furthermore,
most (94.8%) of the cholesterol-supplemented, alcohol-exposed embryos
(n=58) appeared grossly normal, unlike 83.3% of the alcohol-exposed,
unsupplemented embryos (n=96) that had FASD-like phenotypes (FIG. 5C).
Thus, cholesterol supplementation rescues alcohol inhibited Shh
signaling, and prevents alcohol-induced defects at the molecular,
cellular, and developmental levels.

[0077]In a number of previous studies, researchers have used zebra fish to
determine alcohol-related effects on development.55-59 Applicant has
extended these results by using this model to identify a novel molecular
mechanism that may be responsible for alcohol's teratogenic effects,
namely, alcohol-induced inhibition of the cholesterol modification of
Sbh, which subsequently inhibits Shh signal transduction; inhibition of
this pathway appears to play the key role in the development of FASD
pathogenesis.

[0078]As zebra fish lack placentas and develop ex utero, and alcohol
dehydrogenases60,61 are not expressed in embryos at the time exposed
to alcohol (ie from 4-10 hpf), Thus, the metabolites generated by
oxidation of ethanol are not likely to be a major cause of the induced
phenotypes. Even at very low tissue concentration, alcohol may directly
causes developmental defects, instead of alcoholic metabolites from
maternal resource or the embryo.

[0079]Direct measurements determined a range of alcohol concentration from
0.71-7.4 mM or 0.003-0.034 g dl-1 in fetal tissue under our
experimental conditions. These alcohol concentrations are about 5.9- to
123-fold lower than blood alcohol levels that induce FASD in mice62;
these concentrations are also 4.2- to 153-fold lower than the alcohol
concentrations that induce cell apoptosis63, and retinoic acid
deficiency37 or that have antagonistic effects on growth
factors36. Hence, relatively low concentrations of fetal tissue
alcohol also can induce FASD-like defects. Blood alcohol concentrations
in this range are achieved in a 55-kg female following the consumption of
one 12-ounce beer. This may explain why alcohol is the most common
teratogen responsible for human congenital defects, and suggests that
there is no safe level of alcohol consumption during pregnancy.

[0080]Fetal alcohol exposure impairs hedgehog cholesterol modification and
signaling. The morphological phenotypes induced by alcohol in zebra fish
recapitulate the FASD defects seen in other species. For instance,
similar defects have also been reported in human embryos that have
FASD23,64 microcephaly in 84%, eye problems in 25%, cardiac
developmental defects in 29%, various problems with truncal muscles and
bones, including slack muscles in 58%, swallowing/feeding problems in
20%, hip deformities in 9%, pidgeon chest in 30%, concave chest in 7%,
and spinal dimple in 44%. FASD patients also have craniofacial
abnormalities such as facial anomalies (95%), small teeth (16%), cleft
palate (7%), and overall growth retardation (98%). Thus, it seems that
the ethanol-related developmental defects that applicant has observed in
zebra fish embryos nicely parallel those observed in humans having FAS.

[0081]The evidence presented here and elsewhere39,65 consistently
demonstrates that fetal alcohol exposure inhibits the transcription of
Shh responsive genes. Notably, applicant found that total Shh protein
level in the zebra fish embryonic tissues was not significantly changed
by any of the tested alcohol exposures. However, the treated embryos
exhibited FASD-like phenotypes and unpaired Hh signal transduction,
suggesting that defective Shh signaling is the key factor in the
morphological defects induced by alcohol. Furthermore, applicant has now
shown that the defect in Shh signal transduction is due to disruption of
the post-translational cholesterol modification of Shh. These findings
help to explain why over-expression of Shh mRNA alone does not
consistently rescue alcohol-induced morphologic defects or the decrease
in expression of Shh responsive genes. 38,65

[0083]The mature Shh peptide is doubly lipid-modified, having a
cholesterol moiety at its C terminus4 and a palmitate attachment at
Cys-24 of the N terminus.67 The N-terminal lipid is required for
inducing the differentiation of ventral forebrain neurons.68 In
contrast, in the absence of the N-lipid, the C-terminal lipid-containing
Shh is sufficient to induce mouse digit duplication.69 Mouse mutants
have been created in which Shh lacks cholesterol modification, lacks
palmitoylation, or lacks both types of lipid modification. Functional
analysis of these mutants clearly demonstrated that both types of lipid
modification are essential for regulating the range and shape of the Shh
morphogen gradient during early development.70.71 For future
direction, it remains to be determined whether the alcohol-induced defect
in cholesterol modification influences N-terminal palmitoylation, or Shh
cellular trafficking in lipid rafts, or affects the binding affinity of
Shh to Ptc, or even the gradient shape and content of Shh.

[0084]Overall, applicant has shown that a simple post-translational
modification defect of a key morphogen results in a complex congenital
disease. This new insight into the molecular basis of FASD has
far-reaching implications, and suggests novel prenatal interventions that
might prevent FASD developmental defects.

Caveolin-1 Binds with Shh to Form a Protein Complex

[0085]Applicant has observed that alcohol exposure results in defective
Shh-cholesterol modification and impairs the accrual of plasma membrane
associated Shh, suggesting that alcohol causes a defect in the
intracellular transport of this ligand (Li, 2007). In order to obtain
detailed information on the distribution of Shh within cells, and as a
first step toward determining the mechanisms underlying Shh intracellular
trafficking, applicant used non-ionic detergent protein extraction and
density gradient ultracentrifugation to fractionalize and isolate
cellular proteins. After ultracentrifugation, applicant collected 17
individual gradient fractions (each fraction contained 500 μl), from
the lowest density (at the top of the centrifuge tube) to the highest (at
the bottom of the tube). The distribution of lipid raft proteins was
primarily confined to fractions 4 through 11, as indicated by the
presence of the lipid raft-associated protein, Caveolin-1 (FIG. 6A,
bottom panel). Applicant also found that the distribution of Shh was
confined to fractions 6 through 17; Shh co-localized with Caveolin-1 in
fractions 6 through 11 generated by the density gradient
ultracentrifugation (FIG. 6A, top panel).

[0086]FIG. 6 shows representative western blot analysis of Caveolin-1 and
Shh distribution in density gradient ultracentrifugation fractions for
protein lysates isolated from the rat hepatic stellate cell line HSC 8B
(A). immunoprecipation assays demonstrate that Shh and Caveolin-1
physically interact to form a protein complex (B, C). Equal amounts of
cell lysates were used in the immunoprecipitation assays and expression
levels of the target proteins were confirmed by Western blot analyses
using either anti-Caveolin-1 antibody (B, top pane, line 2) or anti-Shh
antibody (C top panel, line 2). Both Caveolin-1 (B, middle panel and B
bottom panel) and Shh (B bottom panel and C middle panel) were detected
in both anti-Caveolin-1 and anti-Shh antibody immunoprecipitates, but not
in the IgG negative control precipitates (line 1, middle and bottom B and
C).

[0087]Physical co-localization of Shh and Caveolin-1 in the density
gradient may suggest that these two proteins have same similar physical
characteristics or that they functionally interact, however, it does not
necessarily indicate a direct physical interaction between them. Since
Caveolin-1 plays an important role in protein and cholesterol transport
and trafficking, a direct interaction between Caveolin-1 and Shh is an
intriguing possibility. In order to establish whether these proteins
physically interact to form a protein complex, applicant performed a
series of immunoprecipitation assays on total protein isolated from the
rat hepatic stellate cell line, HSC8B, which expresses high levels of
Shh. To determine whether there is a direct physical interaction between
these two proteins, applicant used an anti-Caveolin-1 antibody in an
immunoprecipitation assay of HSC8B cell lysates, followed by Western
blots analyses using anti-Caveolin-1 and anti-Shh antibodies. Applicant
confirmed equal loading of the precipitated proteins using the
anti-Caveolin-1 antibody (FIG. 6A, top panel). As expected, the
anti-Caveolin-1 antibody precipitated the 22 kDa Caveolin-1 protein (FIG.
6B, middle panel, line 2). Furthermore, it precipitated the mature 20 kDa
Shh ligand (FIG. 6B, bottom panel, line 2), indicating that Caveolin-1
physically associates with Shh. In the negative control, IgG antibody was
unable to precipitate either Caveolin-1 or Shh (FIG. 6, line 1, middle
and bottom panel). Moreover, applicant confirmed the interaction between
these two proteins by using an anti-Shh antibody in an
immunoprecipitation assay of the HSC8B cell lysate. The conditions used
in this experiment paralleled those used in the previous
immunoprecipitation assay; in this case, equal protein loading was
confirmed using the anti-Shh antibody (FIG. 6C, top panel). Applicant
found that both Shh (FIG. 6C, middle panel) and Caveolin-1 (FIG. 6C,
bottom panel) are immunoprecipitated from HSC8B cell lysates by the
anti-Shh antibody. The fact that anti-Shh and anti-Caveolin-1 antibodies
each co-precipitate Caveolin-1 and Shh from HSC8B cell lysates strongly
suggests that Caveolin-1 interacts with Shh to form a protein complex in
this Shh producing cell line.

Alcohol Specifically Decreases the Formation of the Caveolin-1/Shh Complex
in Lipid Rafts

[0088]The observation that Shh directly interacts with the lipid raft
protein Caveolin-1 raises an interesting question regarding previous work
in which applicant showed that alcohol exposure does not affect total Shh
levels, but instead results in defective Hh signal transduction by
causing a dose-dependent decrease in the concentration of
cholesterol-modified, mature Shh ligand at cell plasma membranes (Li,
2007). To determine whether this decrease in plasma membrane-associated
Shh ligand is specifically linked to the interaction between Caveolin-1
and Shh in lipid raft microdomains, applicant investigated the affect of
alcohol exposure on the co-localization of Caveolin-1 and Shh in density
gradient fractions. First, as described in FIG. 6, cellular proteins
isolated by non-ionic detergent resistant cellular extraction were
fractionalized by density gradient ultracentrifugation. Lipid raft
associated proteins were present in fractions 4 through 11, as indicated
by the presence of Caveolin-1 (FIG. 6A, middle panel and FIG. 9A, second
panel); applicant specifically used fractions containing lipid raft
associated proteins (fractions 7-9) for use in immunoprecipitation assays
with an anti-Caveolin-1 antibody. HSC8B cells were exposed to various
concentrations of alcohol (0, 0.3, 0.5, 0.6, and 0.8% w/v corresponding
to 0, 55, 81, 109 and 136 mM) for two hours prior to protein extraction
and density gradient ultracentrifugation. Fractions 7 through 9 from the
density gradient were pooled and used in the immunoprecipitation assay.

[0089]Equal loading of proteins for use in these assays was confirmed by
Western blot analysis using the anti-Caveolin-1 antibody (FIG. 7A). In
these immunoprecipitation assays, applicant determined that the amount of
Shh found in the Caveolin-1 containing lipid raft fractions (fractions
7-9) decreased in an alcohol dose-dependent manner (FIG. 7C); the alcohol
treatments did not affect the amount of Caveolin-1 in the lipid raft
fractions (FIG. 7B). IgG antibody, which was used in negative control
immunoprecipitation assays, did not precipitate either Caveolin-1 or Shh.
In FIG. 7, Immunoprecipitation assays demonstrate that alcohol exposure
decreases the amount of Shh in Caveolin-1/Shh complexes located in lipid
raft fractions. Using non-ionic detergent resistant protein extraction,
followed by sucrose gradient centrifugation, lipid rafts and their
associated proteins were isolated from HSC 8B cells treated with various
alcohol concentrations (alcohol concentration w/v: 0.3%, 0.5%, 0.6% and
0.8% for 2 hours) and from untreated cells. Anti-Caveolin-1 antibody was
used to immunoprecipite proteins from lipid raft preparations; equal
amounts of protein were ensured by Western blot analysis using the
anti-Caveolin-1 antibody (A), and the immunoprecipitates were probed with
both anti-Caveolin-1 (B) or anti-Shh antibodies (C). The amount of
Caveolin-1 protein in the lipid rafts was not affected by alcohol
exposure (B); however, alcohol exposure decreased the amount of
Caveolin-1-associated Shh in a dose-depended manner (C).

[0090]We also used immunohistochemistry to evaluate the effect of alcohol
on Caveolin-1/Shh complex formation. In FIG. 8, immunohistochemistry
revealed co-localization of Caveolin-1 and Shh. In untreated hepatic
stellate cells, Caveolin-1 (A, green) and Shh (B, red) co-localized in
the cytoplasm, and particularly in the cell plasma membrane (C, yellow,
indicated by arrows). When cells were exposed to 0.6% (w/v) alcohol for
30 minutes, Shh levels were not affected; however, the amount of Shh
co-localizing with Caveolin-1 at the plasma membrane dramatically
decreased. Double-staining HSC 8B cells with anti-Caveolin-1 antibodies
(FIG. 8A, green) and anti-Shh antibodies (FIG. 8B, red) revealed a
punctate, salt-and-pepper, co-localized distribution of Shh and
Caveolin-1 in the cytoplasm, particularly at the plasma membranes (FIG.
8C, yellow as indicated by arrows). Exposure of HSC 8B cells to 0.4%
(w/v) (FIGS. 8D-F) and 0.8% (w/v) (FIG. 8G-I) alcohol for thirty minutes
did not produce noticeable changes in either Caveolin-1 (FIGS. 8D and 8G,
green) or Shh (FIGS. 8E and 8H, red) levels; however, the amount of
punctate co-localized particles dramatically decreased in an alcohol dose
dependent manner, nearly disappearing from the cell plasma membranes at
high alcohol concentrations (FIGS. 8F and 8I, yellow). Both biochemical
and immunohistochemical analyses indicated that Caveolin-1 and Shh form a
protein complex, and showed that this complex accumulates in a lipid raft
domains in plasma membranes. Alcohol exposure disrupts the formation of
the Caveolin-1/Shh complex and leads to decreased levels of Shh in plasma
membranes, particularly in the lipid raft structures. These observations
suggest that alcohol exposure causes a defect in the secretion and
transport of the Shh ligand.

Alcohol Exposure Causes Defective Transport of Shh into the ER and Leads
to Accumulation in the Golgi Compartment

[0091]In order to begin delineating the detailed molecular mechanisms that
underlie the deleterious effect of alcohol on Shh transport in
ligand-producing cells, applicant first compared the distribution of Shh
among the cellular proteins fractionated by density gradient
ultracentrifugation to the distribution of lipid rafts (Caveolin-1), and
Golgi and ER compartment markers.

[0092]In FIG. 9, alcohol disturbs Shh co-localization with Caveolin-1 in
lipid rafts and causes Shh to accumulate in Golgi organelles. To
facilitate analysis of the distribution of Shh, cellular proteins were
fractionally isolated by density gradient ultracentrifugation; its
distribution was compared to the distribution of a lipid raft protein
(Caveolin-1), ER and Golgi compartment markers. In untreated cells,
Western blot analysis indicated that Shh protein was located in density
gradient fractions 7 to 17 (FIG. 4A, top panel); fractions 7 to 11
contained lipid raft fractions as indicated by the presence of Caveolin-1
(FIG. 4A, middle panel); fractions 12 to 17 correspond to the Golgi/ER
compartments as indicated by the presence of the Golgi marker (FIG. 4A,
third panel). In HSC 8B cells exposed to 0.8% w/v alcohol for 30 minutes,
the distribution of Shh shifted out of the Caveolin-1/lipid raft and
smooth ER fractions, and was restricted to density gradient fractions 12
to 17, which correspond to the Golgi-associated protein and rough ER
fractions (FIG. 4B, top panel).

[0093]In protein extracts isolated from cells that were not exposed to
alcohol, Western blot analyses indicated that Shh was broadly distributed
in the density gradient, from fraction 7 through 17 (FIG. 9A, top panel);
in these extracts, lipid raft associated proteins were distributed in
fractions 7 through 11, as indicated by the presence of Caveolin-1 (FIG.
9A, second panel). Golgi-associated proteins were present in fractions 12
through 17 (FIG. 9A, third panel); and endoplasmic reticulum-associated
proteins were located in fractions of 6 through 9 and 16 and 17 (FIG. 9A,
bottom panel), as demonstrated by the presence of specific Golgi/ER
markers. In protein extracts isolated from HSC 8B cells that were exposed
to 0.6% alcohol (w/v, 109 mM) for 30 minutes, the density gradient
distribution of Shh was restricted to fractions 12 through 17 (FIG. 9B,
top panel); these fractions contain proteins associated with the Golgi
and rough ER (FIG. 9B, third panel), indicating that alcohol exposure
shifts Shh distribution away from the lipid raft-containing (FIG. 9B,
middle panel) and smooth ER compartment fractions (FIG. 9B, bottom
panel).

[0094]In FIG. 10, alcohol is shown to disrupt Shh entry into ER
compartments and causes Shh to accumulate in Golgi organelles. Cells were
prepared for immunohischemistry analysis by co-staining with anti-Shh
antibody and anti-ER or anti-Golgi marker antibodies or both. In
untreated cells, the ER marker (PID) (A, green) and Shh (B, red)
co-localized in the cytoplasm as punctate particles (C, yellow). However,
in cells exposed to 0.6% (w/v) alcohol for one hour, although the
expression levels of the ER marker (D, green) and Shh (E, red) were
unchanged, the punctate, polarized distribution pattern of Shh was not
detected; instead applicant observed a defused homogeneous distribution
of Shh (E and F). G-I: no alcohol exposure; J-L: 0.6% (W/V) alcohol
exposure for 1 hour. H and K: anti-Shh antibody staining (red); G and J:
anti-Golgi marker antibody staining (green) and I, L: merged
corresponding images (yellow). Alcohol treatment did not affect the Shh
expression level or its distribution in the Golgi compartment.

[0095]To further elucidate the defects that alcohol exposure causes in Shh
secretion, applicant used confocal microscopy to examine cells stained
with anti-Shh antibodies and antibodies against either Golgi or ER
compartment markers. In untreated cells, the ER marker (identified using
an anti-PID antibody) (FIG. 10A, green) and Shh (FIG. 10B, red)
co-localized in the cytoplasm in punctate particles (FIG. 10C, yellow);
however, when HSC 8B cells were exposed to 0.6% (w/v) alcohol for one
hour, although the ER marker (FIG. 10D, green) and Shh (FIG. 10E, red)
levels were not significantly effected, rather than a punctate, polarized
distribution pattern, applicant observed a diffuse, homogeneous
distribution pattern for Shh (FIG. 10F). Under the same experimental
conditions, applicant determined whether Shh (FIGS. 10E and 10K, red) and
the Golgi marker (FIGS. 10G and 10J, green) co-localized by overlaying
the corresponding images (FIGS. 10I and 10L, yellow) and found that
alcohol treatment did not significantly alter Shh distribution in the
Golgi compartment. Hence, the observation that Shh accumulates in the
Golgi with no specific entry into the smooth ER indicates that the
primary defect in Shh transport caused by alcohol exposure occurs in
trafficking between the Golgi and the smooth ER compartments. Previously,
applicant demonstrated that alcohol exposure inhibits the
post-translational modification of Shh by cholesterol, decreases the
amount of the mature Shh ligand that is associated with the cell
membrane, and leads to a spectrum of defects in our zebra fish model that
phenocopies the defects observed in patients having Fetal Alcohol
Syndrome (Li, 2007). In this study, applicant has investigated the
mechanism underlying alcohol inhibition of the Hedgehog signal
transduction in further detail. Applicant has determined that alcohol
exposure leads to defective intracellular transport of the Shh ligand by
inhibiting the ability of Shh to form a complex with Caveolin-1,
preventing its translocation to the plasma membrane in lipid raft
domains.

Alcohol Exposure Disrupts Shh Secretion

[0096]Alcohol exposure inhibits Shh in the Golgi from entering the smooth
ER and, therefore, prevents the formation of a Shh/Caveolin-1 protein
complex in lipid raft domains, which leads to defective transport of the
Shh ligand to the plasma membrane and results in Shh accumulation in the
Golgi compartment. Applicant deduced that defective intracellular
transport of Shh can lead to decreased secretion of the Shh ligand into
the extracellular matrix. To confirm this hypothesis, applicant focused
on the effect of alcohol exposure on Shh accumulation in the medium of a
cultured Shh producing cell line, HSC 8B. Applicant analyzed proteins
collected from the HSC 8B culture medium for Shh ligand content using two
independent methods: Western blot analysis and Elisa assay. When the
density of HSC 8B cells in culture dishes reached 75% confluence,
applicant replaced the culture medium with fresh medium containing serum
replacement and various concentrations of alcohol (0, 0.15, 0.3, 0.6 and
0.8% w/v corresponding to 0, 25, 55, 109 and 136 mM). The cultures were
incubated for an additional 3 hours, and culture medium was then
harvested and concentrated for protein isolation. As shown in FIG. 11A,
Western blot analysis of proteins that accumulated in the culture medium
indicated that alcohol inhibits Shh secretion in a dose-dependent manner;
Elisa assays indicated similar results. In detail, exposure of HSC 8B
cells to 0.3%, 0.6% and 0.8% (55, 109 and 136 mM) alcohol concentrations
corresponded to 1.2, 4.2 and 5.5 fold decreases in Shh secretion (FIG.
11B). Our results delineate a molecular mechanism for alcohol inhibition
of the hedgehog signal transduction pathway in which alcohol inhibits
cholesterol-modification of Shh, which hinders Shh binding to Caveolin-1,
prevents its entry into the smooth ER, and disrupts its subsequent
transport to the plasma membrane in lipid raft domains, resulting in
decreased Shh secretion into the extracellular matrix.

Cholesterol for Stem Cell Nutrients

[0097]Cholesterol and its derivatives are nutrients for maintaining
physiological function of Hedgehog (Hh) dependent stem cells in embryo
and adult tissue. Hedgehog ligands and receptor are expressed in the
liver. Hh-responsive cells exist in early embryonic stages, but rarely in
adult normal liver.

Transgenic Zebra Fish with Labelled Mature Liver Cells

[0098]Transgenic zebra fish (LFABP-GFP) express GFP in mature hepatocytes
and cholangiocytes. A transgenic zebra fish, LFABP-GFP, is the model used
for searching for hepatic stem cells. In this transgenic line, all mature
hepatocytes and cholangiocytes are labeled with GFP protein via
expression driven by the liver fatty acid binding protein (LFABP)
promoter.

[0099]In FIG. 11, alcohol exposure disrupts Shh secretion into the
extracellular matrix. Medium from the cultured HSC 8B cell line was
collected and concentrated for use in Western blot analyses (A) and Elisa
assays (B). Exposure to various alcohol concentrations (0.15%, 0.3%, 0.6%
and 0.8% V/V corresponding to 25, 81, 109, and 136 mM) for 3 hours
inhibited Shh secretion in a dose-dependent manner. * indicates a
p<0.05.

[0101]GFP expression in these cells is initiated on embryonic day 2 (FIG.
12) and is maintained throughout the entire life span. As shown in FIGS.
12B and 12C, the GFP labeled liver can be clearly observed in the living
adult zebra fish (6.5 months) under fluorescence microscopy. High levels
of GFP expression can be seen in whole liver surgically removed from the
fish, blood vessels excepted. Using anti-GFP antibody, immunohistological
analyses of liver sections revealed that mature hepatocytes and
cholangiocytes in these fish express GFP; moreover, this GFP expression
pattern recapitulates the endogenous expression pattern of LFABP that is
expressed in hepatocytes and cholangiocytes, but not in nonparenchyma
(FIGS. 12E and 12F). Fluorescence activated cell sorting (FACS) was used
to separate liver cells into two populations. The GFP positive population
contained mature hepatocytes and cholangiocytes. The putative hepatic
stem cells were located in the GFP negative fraction. Since GFP is
controlled by a specific gene (LFABP) promoter, the GFP expression is
restricted in differentiated hepatocytes and cholangiocytes.

[0102]This unique feature provides an ideal means for monitoring whether
GFP negative cell differentiate into GFP positive cells in an ex vivo
transplantation model. When GFP negative/Ptc positive cells are
transplanted into a wild type fish, the emergence of GFP positive cells
in the recipient liver indicates that some of the donor cells have
differentiated, becoming mature hepatocytes or cholangiocytes or both.
Therefore, it also allows dynamic monitoring of the differentiation
process of transplanted cells in the recipient fish.

Transgenic Zebra Fish

[0103]In the transgenic zebra fish, nonparenchymal Ptc positive cells
(GFP-/Ptc+), which comprise 0.05% of the adult liver cell population, are
morphologically different than mature hepatocytes. Ptc positive (Ptc+)
cells were isolated from the GFP negative (GFP-) fraction of the
transgenic liver cell population. First, the LFABP-GFP liver was
perfused, and then FACS were used to sort out GFP- cells twice from GFP+
cells. Second, GFP- fraction was immunostained with anti-Ptc antibody,
followed by secondary antibody incubation which is conjugated with
Rhodamine florescence (FIG. 13A). Therefore, the GFP-/Ptc+ cells (that
have no green fluorescent/high red fluorescent) were isolated by another
round of FACS.

[0104]In FIG. 13, cell cytometry is used to isolate GFP-/Ptc+ cell from
LFABP-GFP liver (A). Gene expression analysis by real-time quantitative
RT-PCR shows that Ptc-antibody sorted GFP- cells are enriched with
transcripts of Ptc and Aldh2, but not Shh (B).

[0105]In summary, GFP-/Ptc+ cells comprise about 0.05% of the whole liver
cell population. Compared to mature hepatocytes, which are about 12-18
μm in diameter, GFP-/Ptc+ cells are small, having diameters of about
4-6 μm. Real time quantitative RT-PCR analysis (FIG. 13B) confirmed
that these cells express high levels of Ptc mRNA, 27-fold higher than
mature hepatocytes. Another stem cell marker gene, Aldh2, is enriched in
GFP-/Ptc+ cells (20-fold higher than expression levels in GFP+ mature
hepatocytes and cholangiocytes).

[0107]The GFP-/Ptc+ cells (FIG. 14A) are difficult to culture in typical
culture medium; no cell divisions occur in the first two weeks in
culture, and gradually the cells die. After trying several different
culture conditions, these cells were found to prosper in collagen IV and
laminin coated culture dishes incubated at 28.5° C. A
hepatocyte-inducing medium was formulated that contains 100 ng/ml FGF1,
20 ng/ml FGF4 and 50 ng/ml HGF. In this medium, GFP-/Ptc+ cells start to
express GFP, indicating that the cells have differentiated into
hepatocytes or cholangiocytes or both.

[0108]After culturing these cells for 5 days, cells started to express GFP
and transformed like hepatocyte (FIG. 14B). After 14 days in culture,
colonies were formed in which cells in the center of the colony express
GFP (FIG. 14C); a band of GFP negative cells, 7-9 cells wide, surround
the GFP positive cells. These GFP negative cells at the edge of the
colonies become GFP positive after an additional 2-3 days, but new GFP
negative cells emerged (or migrated) to form a new GFP negative band of
cells at the edge of the colonies (FIGS. 14C and 14D).

[0110]To determine whether these small-sized GFP-/Ptc+ cells are
multipotent hepatic stem cells, the fate of these cells was investigated
by transplanting them into wild type zebra fish and medaka. The day
before transplanting the cells, the recipient zebra fish were injected
with Tunicamycin, a protein translation inhibitor, to induce extensive
liver injury and hepatocyte death. One hundred donor cells (GFP-/Ptc+)
were injected intraperitoneally into a recipient wild fish. One week
after transplantation, GFP expression was observed in recipient fish when
examined under fluorescent microscope. Frozen sections of the recipient
liver showed that GFP positive cells had repopulated the liver.
Furthermore, GFP monoclonal antibody staining revealed that the donor
cells were undergoing very rapid proliferation and differentiation into
biliary ductular epithelial cells and some into hepatocytes (FIGS. 15E
and 15F). One month after transplantation, the recovered liver contained
many GFP positive (FIG. 15L) hepatocytes (10%) that were descendents of
the transplanted GFP-/Ptc+donor cells (FIGS. 15G and 15H). This clearly
illustrates, as was seen in cell culture, that when transplanted into
adult zebra fish with previously injured livers, Ptc positive
nonparenchymal cells can differentiate into hepatocyte- and
cholangio-like cells. These results provide a solid basis for testing the
overall hypothesis of this project: that Hh signaling may regulate
self-renewal, expansion and differentiation of stem cells in the adult
liver.

[0111]Alcohol disrupts Shh protein (red) and lipid raft (green)
co-localization (yellow) and results in Shh transportation and secretion
defects as seen in FIG. 16. A-C are the controls without alcohol
treatment; D-E treated with 0.25% (V/V) alcohol for 5 minutes; G-I
treated with 1.0% alcohol for 1 hour. Lipid raft is labeled by green
fluorescent in confocal images of A, D and G; Shh protein is shown by red
fluorescent in B, E and H. Merged images are C, F and I in which the
yellow signals indicates of the co-localization of Shh and lipid rafts.

[0112]In order dissect the detail mechanism of alcohol induced hedgehog
signaling defect, the Shh protein transportation in cell was monitored
(FIG. 16). A hepatic stellate cell line, HSC8B, from adult rat liver, was
chosen as the model to study the dynamic changes of Shh trafficking and
co-localization of lipid rafts. Lipid raft was labeled with Vybrant Lipid
Raft Labeling Kits (Molecular Probe, Catalog number V34403). This
labeling system provides convenient, reliable and extremely bright
fluorescent labeling of lipid rafts in live cells.

[0113]Lipid rafts are detergent-insoluble, sphingolipid- and
cholesterol-rich membrane microdomains that form lateral assemblies in
the plasma membrane. It uses the nature affinity of a bacterial toxic
protein, cholera toxin subunit B (CT-B), that secreted from Vibrio
cholerae bacterium and can specifically binds a constitutional lipid of
lipid raft. The Vybrant Lipid Raft Labeling Kits provide the key reagents
for fluorescently labeling lipid rafts in vivo with bright and extremely
photostable ALEXA FLUOR dyes. Live cells are first labeled with the
green-fluorescent Alexa Fluor 488 (or other color dyes) conjugates of
cholera toxin subunit B (CT-B). This CT-B conjugate binds to the
pentasaccharide chain of plasma membrane ganglioside GM1, which
selectively partitions into lipid rafts. An antibody that specifically
recognizes CT-B is then used to crosslink the CT-B labeled lipid rafts
into distinct patches on the plasma membrane, which are easily visualized
by fluorescence microscopy. When liver stellate cells were treated with
alcohol concentration at 0.25% v/v) just for 5 minutes, the
co-localization of Shh (red) and lipid raft (green) is disrupted (less
yellow comparing to no alcohol treated), when alcohol concentration
increased to 1% for one hour, the Shh protein expression level was not
affected, but almost all of the Shh protein was accumulated inside of the
liver stellate cells, no association with lipid raft or cell membrane.

[0114]Therefore the bottom line of the alcohol pathological mechanism has
been found that alcohol inhibited cholesterylation of Hedgehog protein,
without lipid anchor, the Shh protein could not be associated with lipid
raft and failure for secretion; therefore these transportation defects
lead to this morphogen gradient abnormality that results in development
problems such as fetal alcohol syndrome, as well as liver cirrhosis.

[0115]Furthermore, in adult stem cell in many organs, it has been
demonstrated that Hedgehog pathway plays a major role for participating
tissue regeneration and repairing. These stem cells are found in brain,
skin and digestive system. Alcoholism speeds aging process and induces
liver damage, even liver cirrhosis. Providing cholesterol and cholesterol
derivatives may hold a key to maintain adult stem cell function and
prevent alcoholic aging and diseases, such as cirrhosis.

Summary of Studies

[0116]1. Based on applicant's previous work on Ptc-lacZ transgenic mice,
applicant noted that the numbers of Ptc positive cells decreased
dramatically in mice after embryonic Day 11 during the hepatocyte
differentiation. In adult mice, all mature hepatocytes lack Ptc
expression, but a very few positive Ptc expressing cells are located near
the bile ductular plate. Using sucrose gradient centrifugation, applicant
found Ptc positive cells in the nonparenchymal fraction of the liver cell
population. More importantly, Ptc positive cells can be induced to
proliferate and differentiate under pathological stimulation. Bile
ductule ligation leads to a remarkable increase in the proliferation of
Ptc positive cells; furthermore, some of these cells are progenitors of
the oval cell lineage. These results strongly suggest that a cell
membrane protein, the Hh receptor, Ptc, may be useful to identify and
isolate quiescent adult hepatic stem cells from adult livers.

[0117]2. Given that pathological stimulation induces Ptc positive cell
proliferation and differentiation, Ptc positive cells were isolated from
adult livers. Using a unique transgenic zebra fish model, Ptc positive
cells were purified from the nonparenchymal fraction of the adult liver
cell population. These relatively small Ptc positive nonparenchymal cells
can be induced to differentiate into hepatocytes and cholangiocytes when
transplanted into adult zebra fish having previously injured livers.

[0118]3. Liver regeneration is necessary to repair damaged livers,
including livers damaged by chronic consumption of alcohol. Accumulating
evidence suggests that the pathological mechanism for alcohol-induced
defects may involve in impaired Hh signaling. Fetal alcohol
administration results in similar abnormalities to those seen in animals
having Hh signaling defects or cholesterol metabolic defects. Hh
signaling controls the development of the organs or tissues that are also
the most vulnerable targets in Fetal Alcohol Syndrome. In zebra fish,
applicant has found that alcohol can inhibit Hh signaling by disrupting
cholesterol homeostasis, impairing cholesterol-Shh modification and Shh
transportation in zebra fish embryo and rat adult liver cell.
Supplemental cholesterol rescues cholesterol modification of Shh,
restores Hh signaling, and prevents alcohol-induced developmental
defects.

[0119]The consumption of alcohol is on the rise, especially in women.
Overall, its effects on the fetal development are more harmful than those
attributed to cocaine, heroin or marijuana. In the United States, FAS is
the leading cause of mental retardation and congenital defects,
surpassing even spina bifida and Down's syndrome. Approximately 50,000
children are born with alcohol-related defects in the U.S annually.
Despite prenatal education and general public awareness, one out of five
pregnant women is believed to consume alcohol during pregnancy. Moreover,
45% of women who consumed alcohol also reported that they did not learn
of their pregnancy until after the fourth week of gestation. As a result,
the cost of alcohol-related birth defects is an estimated US$ 9.7 billion
annually. Though extensive research work is continuing on FAS, little
conclusive evidence is available beyond the documented fact that alcohol
is harmful to the developing fetus.

[0120]Detecting alcohol use amongst pregnant women is an important step
toward preventing alcohol-related birth defects. Since maternal alcohol
use is under-reported and identification of alcohol-exposed newborns is
often difficult in the absence of severe FAS defects, a biomarker that
could detect alcohol use during pregnancy would aid in earlier
identification and intervention for pregnant mothers and affected
infants. More importantly, early intervention for affected children
before the age of six may reduce the incidence of anti-social behavior
later in life.

[0121]Our observation suggests a novel mechanism for the teratogenic
effects of alcohol via alterations in cholesterol metabolism. Even at
very low concentrations of alcohol exposure, applicant observed
consistent and dramatic decreases in cholesterol ester levels and in the
ratio of cholesterol ester-to-total cholesterol in zebra fish embryos.
Furthermore, applicant also find impaired signaling in the Hedgehog
pathway, which plays a key role in the embryonic development of numerous
organs and structures that are vulnerable to prenatal alcohol exposure.
This altered signaling appears to be due to defects in the cholesterol
modification of Hedgehog ligand. To confirm our findings in a mammalian
system, applicant has chronically fed mice an alcohol diet and find
similar aberrations in cholesterol homeostasis. These mechanistic studies
provide a solid indication that focusing on metabolic profile analysis of
free fatty acids and cholesterol will lead to a new set of biomarkers for
alcohol exposure.

[0122]Based on our previous data, our goal is to identify a biomarker
signature that detects maternal and prenatal alcohol exposure by
fingerprinting metabolic intermediates, such as cholesterol chemically by
clinic test or physically by Raman Spectroscope.

[0123]Raman Spectroscope: When light passes through matter, most photons
continue in their original direction but a small fraction are scattered
in other directions. Light that is scattered due to vibrations is called
Raman scattering or the Raman Effect. The difference in energy between
the incident photon and the Raman scattered photon is equal to the energy
of a vibration of the scattering molecule. A plot of intensity of
scattered light versus energy difference is a Raman Spectrum. The
measurement of the identity and intensity of Raman Spectrum can
specifically identify molecules and their concentration in a complicated
system. This is the physiochemical basis of the Raman spectroscope.

[0124]Our previsions data (FIG. 3) demonstrate that alcohol exposure
during the late blastula-gastrula stage causes a dose-dependent reduction
in membrane-associated Shh. Given that esterification of Shh by
cholesterol drives its membrane localization, these results suggest that
alcohol exposure reduces cholesterol ester formation. To screen for this
possibility, applicant measured cholesterol ester content in whole embryo
extracts that were exposed to various doses of alcohol for 3 h during the
late blastula-gastrula stage. Applicant next tested whether alcohol
exposure impairs general sterol homeostasis during gastrulation by
measuring cholesterol levels in whole embryo extracts. In a dose-related
fashion, alcohol exposure resulted in a decrease in the total cholesterol
content of embryos (FIG. 4). This was mostly due to reductions of
cholesterol esters, very minor from the reduction of the free
cholesterol.

[0125]These trends are also seen in the data collected from a chronic
alcohol administration in mice model. The ratio of cholesterol ester to
total cholesterol in plasma is significantly decreased in the alcohol
treated group by comparing both control groups (p<0.001) (FIG. 17A).
In FIG. 17, alcohol exposures disrupt free cholesterol/cholesterol ester
balance and transport in embryos. A. Chronic alcohol feeding decreases
the ratio of cholesterol ester to total cholesterol. B. Filipin staining
showed a significant reduction of free cholesterol in embryo body; Oil
red O staining identified cholesterol ester remarkable decrease in embryo
yolk.

[0126]All of the maternal free cholesterol is deposited in the embryonic
yolk. Upon esterified reaction, then the cholesterol ester can be
transported from the embryonic yolk to the body. In order to investigate
whether there is esterification and transportation defect of cholesterol
in alcohol treated embryos, applicant further determined the distribution
of free cholesterol and cholesterol ester between embryo body and yolk by
two chemicals that can differentiate the free cholesterol and cholesterol
ester. The fluorescent molecule, Filipin can selectively bind to free
cholesterol, but not cholesterol ester; in other hand, the dye of oil red
O identifies neutral lipids molecules such as cholesterol ester, but not
polarized free cholesterol. First, applicant quantitatively analyzed the
free cholesterol by whole mount in situ filipin staining. In situ
quantitative density analysis of filipin-free cholesterol staining showed
that free cholesterol decreased differentially in alcohol-exposed
embryos, showing a large decrease in cholesterol concentration in the
embryonic body, but not in the embryonic yolk. Second, oil red O staining
revealed a dramatic decrease of cholesterol ester in the embryonic yolk,
with little or no change in its concentration in the embryonic body (FIG.
17B). The differential spatial changes in the concentrations of the
different forms of cholesterol indicate that inhibition of cholesterol
esterification by alcohol may lead to deficient cholesterol
transportation from the yolk to the body (tissues), resulting in
hypocholesteromia in embryonic tissues, and subsequently defective Shh
cholesterol modification.

[0127]Applicant has developed "Multimodal multiplex multi-wavelength"
Raman spectroscopy. This system achieves uniquely high optical throughput
and fluorescence rejection for detecting alcohol in tissue as well as
tracing alcohol exposure induced cholesterol signature changes. The
sensor, a combination of spatially coded detection optics and spectrally
coded excitation sources to get the Raman spectrum of alcohol in tissue
(FIG. 18A).

[0129]The plot shows the correlation between measurement principle
spectral component amplitudes and concentration of alcohol. The measured
results were excellent in accuracy for approximately 10% to 0.01% of
tissue alcohol concentrations (FIG. 18B). Applicant has applied the Raman
system to test cholesterol in zebra fish embryos. A signature change in
cholesterol Raman spectrum has been found that it is related to embryos
exposure to alcohol in the Raman spectrum region 1600-1000 cm-1
(FIG. 18C). This alcoholic Raman spectrum is dramatically changed is the
increase of the peaks intensity around 1470, and around 1300 cm-1 in
which the cholesterol peak is clearly decreased under alcohol influence.
Another important difference is the fine Raman features from 1000 to 1200
cm-1, where clear peaks can be detected in alcohol treated embryos.
This change of cholesterol Raman spectrum was also unique by comparing
other two known cholesterol homeostasis inhibitors: Tomxafin (inhibiting
cholesterol esterification) and AY9944 (inhibiting cholesterol
biosynthesis). The signatures of these two drugs related Raman spectrum
are also having far clinic impact for diagnosis and monitoring
cholesterol defects caused by these drugs.

[0130]Cholesterol derivative components rescue alcohol inhibited Hedgehog
signaling activity in rat liver stellate cells. Hepatic stellate cells,
also known as Ito cells, are found in the perisinusoidal space (a small
area between the sinusoids and hepatocytes) of the liver. The stellate
cell is the major cell type involved in liver fibrosis, which is the
formation of scar tissue in response to liver damage. In normal liver,
stellate cells are described as being in a quiescent state. Quiescent
stellate cells represent 5-8% of the total number of liver cells.
Different environmental factors and disease caused liver injury (such as
alcohol exposure) can be activated. The activated stellate cell is
responsible for secreting collagen scar tissue (fibrosis), which can lead
to cirrhosis.

[0131]The Gli-luciferase reporter assay was performed in duplicated
experiments of a rat hepatic stellate cell line, 8H. Briefly, 5H cells,
were grown in DMEM medium supplemented with 10% fetal bovine serum and
penicillin and streptomycin (100 U/ml). At 40-50% confluency, cells were
transfected with Gli-BS-Firefly luciferase plasmid (60 ng nl-1) and
Renilla luciferase plasmid (60 ng nl-1, pRL-TK, Promega)
(concentration ratio 10:1) by using FuGENE 6 Transfection Reagent (Roche
Applied Science). Twenty-four hours later, the medium was replaced with
medium containing different concentration of alcohol. Two hours later,
the medium was added to different drugs (20α-OHC, 22(S)-OHC, 25-OHC
and cholesterol). After 16 hrs, the cells were washed three times in
ice-cold PBS, and then were assayed by Dual/Luciferase Reporter Assay
System (Promega). Activity of the Firefly luciferase reporter was
normalized to the activity of a Renilla luciferase internal control for
transfection efficiency.

[0132]Alcohol treatments (0.25, 0.5, 0.75 and 1.0% v/v) decrease Hedgehog
signaling activates as measured by Gli binding site derived luciferase
activities in hepatic stellate cells when compared to no alcohol
treatment group (A0). After alcohol exposure. 2-3 hours, adding
cholesterol as well as other sterol-like components can rescue the
Hedgehog pathway function back to normal level like the no-alcohol
exposure group (Table II). These tested chemicals and their
concentrations region are described in following Table.

[0133]Cholesterol and cholesterol-like components rescue hedgehog activity
in alcohol treated rat hepatic stellate cell in tissue culture system. In
order to test the rescuing ability to also function in an animal model,
cholesterol and cholesterol-like components were microinjected into zebra
fish embryos (concentration regions are listed in Table II above) at 1-2
cell stage with 0.2 nl of cholesterol and cholesterol derivative. Embryos
were allowed to develop 4.3 hours, and then treated with alcohol for 6
hours as previously described. At 72 hpf embryos were analyzed.

[0135]When zebra fish embryos were treated with 2.0% alcohol for 6 hours
starting from 4.3 hours after fertilization, it causes development
defects such as forebrain truncation, cyclopia and retardation of growth.
Only 15% of these alcohol (2%) treated embryo were developed relatively
normal on gross morphology. On other hand, these 2% alcohol exposure
embryos were supplied with cholesterol and cholesterol-like components,
the percent of gross normal developed embryos increased significantly
reaching to about 80% (See FIGS. 20 and 21). In FIGS. 20 and 21,
cholesterol and cholesterol-like molecules are shown to prevent alcohol
induced embryonic developmental defects. AO: no alcohol exposure; A2: 2%
V/V alcohol exposure. Chol: Cholesterol; 20a OHC: 22a-hydroxycholesterol;
22-OHC: 22-hydroxycholesterol; 25-OHC; 25-hydrocholesterol and Squalene.

Cholesterol Treatment for Adult Tissue

Types of Cholesterol Used

[0136]Cholesterol is a sterol (a combination of a steroid and alcohol) and
a lipid found in the cell membranes of all body tissues, and transported
in the blood plasma of all animals. [0137]1. The chemical name of
cholesterol is
10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,11,12,14,15,16,17-dod-
ecahydro-1H-cyclopenta[a]phenanthren-3-ol. [0138]The chemical formula is
C27H46O. [0139]The chemical structure of cholesterol is
illustrated as following. [0140]The molecular mass is 386.65 g/mol.

[0155]Sigma Company manufactures these cholesterols, most of them being
used for research purposes. Four of these molecules have been tested to
date which have a similarity of cholesterol structure.

Stem Cell Nutrition

[0156]A combination of regular cholesterol and one of the other four
(above) would be safe for over-the-counter supplement for supporting good
t-cell nutrition. To date, cholesterol and other four forms tested have
the ability to maintain function of stem cells that are dependent on
hedgehog signaling in fish embryos and cultured liver cell line. These
functions are rescuing development defects induced by environmental
factors such as alcohol and statins, and function through improving cell
survival ability, proliferation and regeneration ability: Stem Cell
Nutrition.

Nutrient Supplement and Pharmaceutical

[0157]Overall, cholesterol is a natural molecule in our body and intake
cholesterol from food on a daily basis. Cholesterol, even
cholesterol-like substances, can be marketed as a nutrient. All of these
components also have great potential to be developed as new drug.

[0158]These cholesterol compounds may be in conflict with cholesterol
lowering products such as LIPITOR which shows a lot of side effects. The
major use of LIPITOR is lowering cholesterol when it is over 220 mg/dl.
Lowering cholesterol to a very low level will damage stem cells and
related tissue regeneration and aging. Possible side effects of an
increased cholesterol regimen might be high cholesterol level in blood
and tissue. The other cholesterol-like molecules may have a chance to
produce too much stem cells in the body and therefore it may has high
chance to produce tumor.

Liver Treatment

[0159]Cholesterol treatment for liver damage provides more hedgehog
activity for hepatic stellate cells. Oral pill cholesterol form should be
the most common and convenient way. Muscle or vein injection can be used
for special cases.

[0170]Cholesterol treatment is shown herein to prevent overall whole
embryo development defects, but there is not direct evidence for bone
marrow defect benefits. Exogenous supplement cholesterol is the general
approach, taken orally, skin delivered, and by muscle or vein injection.
Cholesterol was delivered by injection as reported herein. Data on bone
marrow effectiveness is yet available. It had been proven how Hedgehog
signaling effects bone marrow stem cell by maintaining or rescuing
hedgehog signaling activity. Some medical conditions for which the OTC or
the prescription cholesterol treatments could help are as follows:
[0171]Leukemia patient with bone marrow transplantation; [0172]Other
cancer patients after chemotherapy or radiotherapy; [0173]Children with
blood stem cell problem.

Neurons in the Brain

[0174]In zebra fish, lab investigation shows how cholesterol prevents
overall whole embryo development defects, specifically for forebrain and
neural tube and neural tube defect. Exogenous supplement cholesterol is
the general approach, taken orally, skin delivered, and muscle or vein
injection. Our data for forebrain and eye defects and their rescuing
approach are presented in the published paper.

[0175]We show dose region of individual cholesterols function on hedgehog
signaling on fish and rat liver cell line. These observations show that
the efficacy order is 25-OHC, 22(S)-OHC, cholesterol, 20α-OHC and
followed with squalene using regular cholesterol. Some medical conditions
for which the OTC or the prescription cholesterol treatments could help
are brain injury, most of chronic brain aging and disease.

Other Organs

[0176]Other body organs which could be helped using the cholesterol and
cholesterol-like therapy treatments of this invention are: [0177]Skin
[0178]Pancreas [0179]G.I. Tract

[0180]While a number of exemplary aspects and embodiments have been
discussed above, those of skill in the art will recognize certain
modifications, permeations and additions and subcombinations thereof. It
is therefore intended that the following appended claims and claims
hereinafter introduced are interpreted to include all such modifications,
permeations, additions and subcombinations that are within their true
spirit and scope.